Alcohol's Effects on Gene Expression.

Altering the expression of specific genes in the brain is one of the mechanisms through which the organism may adapt to chronic alcohol exposure. Several molecular biology methods allow researchers to isolate alcohol-regulated genes from cultured cells or laboratory animals. These techniques have led to the identification of numerous genes whose expression is increased or decreased in cells that have been exposed to alcohol. The protein products of these genes serve a wide variety of functions in normal cells, such as the processing of protein molecules or signal transmission within cells. Preliminary studies are providing insights into the mechanisms through which alcohol may alter the expression of these genes.

C hronic heavy drinkers can tolerate leads to adverse effects in the form of with ing proteins by adding or deleting phos alcohol without exhibiting obvious drawal symptoms. This state is known as phate groups, occur much more rapidly, that signs of impairment at doses that in physical dependence. is, within minutes. Conversely, some changes other people would be incapacitating or even The mechanisms underlying the phe that have been observed after chronic alco fatal. For example, in nonalcoholic people, nomena of tolerance and dependence are hol exposure, such as the "rewiring" of nerve blood alcohol concentrations (BAC's) of 0.6 currently unknown. Increased metabolism cell connections or decreases in the number percent and more usually cause death from of alcohol does not explain why alcoholics' of brain cells, occur too slowly to explain respiratory failure. Chronic heavy drinkers, brains function differently from those of the full range of events seen with tolerance in contrast, frequently survive similar alcohol nontolerant people in the presence of equal and dependence in alcoholics. doses and may even appear sober (Charness alcohol concentrations. The time course for Over the past few years, a number of et al. 1989). This acquired resistance to the ef the development of tolerance and depen researchers have identified several genes fects of high alcohol doses is called tolerance dence does, however, suggest a general whose expression is modified after expo and is one of the criteria used to establish a hypothesis: Changes in the expression of sure of animals or of cells in tissue culture medical diagnosis of alcohol dependence specific genes may account for at least to alcohol. For some of these genes, ex (American Psychiatric Association 1994). some of the dramatic changes evident in pression is increased (Charness et al. 1988; The development of tolerance indicates chronic heavy drinkers' brain functions. Gayer et al. 1991;, where that the brain has adapted to the chronic Behavioral studies in both humans and as for other genes, expression is decreased presence of alcohol. As a consequence of animals have found that tolerance and phy (

REGULATED GENES
The work described in this paper was supported by grants from the National To identify genes whose expression in the starting material, they generally use nerve cells grown and incubated with alcohol in tissue culture or tissue from animals exposed to alcohol. Once a model system is chosen, different technical approaches can be used to characterize the alcoholresponsive genes. Two such approaches are briefly described below.

Study of Candidate Genes
The most straightforward approach to iden tifying alcoholregulated genes is to study genes that already have been isolated and that are known or suspected to contribute to alcohol's effects on the body. Such genes are called candidate genes. By comparing protein production of the candidate genes in cells that have or have not been exposed to alcohol, researchers can determine whether alcohol increases, decreases, or has no effect on the genes' expression.
An example of this strategy is the study of the gene that encodes the enzyme tyro sine hydroxylase (TH). TH catalyzes the first step in the synthesis of a group of neurotransmitters 2 called catecholamines, which includes dopamine and norepine phrine. Alcohol affects the levels of some catecholamines; this process may contribute to the changes in brain function observed after chronic alcohol exposure. To analyze whether alcohol alters the expression of TH, Gayer and colleagues (1991) treated neural cells with alcohol levels correspond ing to BAC's observed in alcoholics. After 3 days, the amounts of both TH messenger RNA (mRNA), an intermediary molecule synthesized during the conversion of genetic information into a protein, and TH protein in the alcoholtreated cells increased almost twofold over the untreated control cells.

Subtractive RNA Hybridization
To discover new genes that may be regula ted by alcohol, researchers must use more sophisticated strategies. One method for identifying unknown genes whose expres sion is increased by alcohol is subtractive RNA hybridization (figure 1). This tech nique uses the mRNA from two batches of neural cells. The RNA from each batch of cells is a mixture, or pool, of mRNA's from many different genes. If both batches of cells are grown under the same conditions, both pools should contain similar amounts of each individual mRNA. If one batch of cells is grown in the presence of alcohol, and if alcohol increases the expression of certain genes, then the RNA pool from the alcoholtreated cells should contain higher levels of the RNA's from the alcohol induced genes than the RNA pool derived from the untreated cells.
To identify the alcoholinduced mRNA's, researchers first copy the RNA pool from the alcoholtreated cells into DNA molecules, which are more stable and easier to handle in the experiment. During this pro cess, the DNA molecules also are labeled with a radioactive "tag" to distinguish them from the mRNA's of untreated cells. Next, both the labeled DNA from the alcohol treated cells and the RNA pool from the untreated cells are mixed with each other in specific proportions. Under certain con ditions, DNA pieces from the alcohol treated cells form pairs with corresponding RNA pieces from the untreated cells. This process is called hybridization. However, because there is an excess of DNA pieces derived from the alcoholinduced mRNA's, not every one of these pieces will be able to find a partner among the mRNA's de rived from untreated cells. Because they are "singlestranded," these unpaired DNA pieces can be biochemically separated from the DNA-RNA hybrids in the mixture. Due to their radioactive tags, these singlestranded DNA molecules then can be used to identify the alcoholregulated genes from which they originated.
Using a similar approach, Miles and colleagues (1994) isolated DNA pieces representing several genes whose expres sion was increased in the presence of alco hol. When the researchers compared these genes to data on other genes stored in a computer database, they found that some of the genes already had been identified in unrelated experiments. For example, two DNA pieces contained the genes for so called molecular chaperones (discussed in more detail below). Alcohol treatment of neural cell cultures significantly increased the mRNA amounts of these two molecular chaperones, GRP78 and GRP94 (figure 2). In contrast, other alcoholregulated genes isolated by subtractive hybridization had not been described before.
The finding that seemingly unrelated genes are regulated by exposure of the cells to alcohol sheds new light on how alcohol affects known functions of nerve cells. The identification of unknown alcoholresponsive genes enables scientists to discover addi tional effects that alcohol has on both in dividual cells and the whole organism.

THE PHYSIOLOGICAL ROLES OF ALCOHOLREGULATED GENES
The alcoholregulated genes and corre sponding protein products that have been identified to date are involved in a wide variety of cell functions. Although a com prehensive review of all these proteins is beyond the scope of this article, some ex amples will be described here in more detail. Table 1 summarizes the functions of ad ditional alcoholregulated genes.

Tyrosine Hydroxylase
As mentioned earlier, the enzyme TH catalyzes a key reaction in the synthesis of a group of neurotransmitters called cate cholamines. One of the catecholamines, dopamine, is involved in regulating motor functions, cognitive functions, emotions, and aggression. Studies in rats found that when the animals received an alcohol dose, the release of dopamine in certain brain areas increased (DiChiara and Imperato 1985). Findings such as these could ex plain why dopamine has been implicated in the sensation of reward associated with abused drugs (Koob 1992), including alco hol, and in the development of tolerance to alcohol (Ritzmann and Tabakoff 1976).
Because TH is essential for the first step of dopamine synthesis, alcoholinduced changes in TH gene expression could contribute to the increased dopamine levels observed after alcohol administration. As described above, experiments with cul tured neural cells found that both TH mRNA and protein levels are elevated after prolonged treatment of the cells with alcohol (Gayer et al. 1991). These findings indicate one mechanism through which al cohol could affect neurotransmitter levels and thus contribute to tolerance develop ment in chronic heavy drinkers. Animal studies on other drugs of abuse (e.g., co caine and amphetamines) also found increased TH expression in brain regions associated with reward sensations (BreitnerJohnson and Nestler 1991).
Researchers now are studying the molecular mechanisms through which alcohol may regulate TH gene expression.
The implications of such studies may have significance beyond the role of the TH gene itself in brain functioning, because the same mechanisms also may apply to many other genes. (See the section "Mechan isms of AlcoholDependent Regulation of Gene Expression," below.)

Molecular Chaperones
Some of the genes whose mRNA increases after alcohol treatment belong to a family of genes that code for proteins called mole cular chaperones. One function of molecular chaperones is, as the name implies, to escort newly made proteins from the point where they are synthesized to their final destination Alcohol can either increase or decrease GABA receptor subunits, depending on the particular subunit and brain region studied.
in the cells (Ellis 1994). The chaperone is vital for the correct functioning of proteins.
Most proteins, after their initial synthesis, undergo chemical modifications that are essential if the proteins are to reach their destinations in or outside the cell and to function properly. The modifications, which usually consist of the addition of sugar molecules or phosphate molecules, occur in specific structures, or organelles, within the cell. Incorrectly modified proteins are degraded immediately or may not function properly if they reach their final destination. The molecular chaperones associate with newly synthesized proteins, guide them to the appropriate organelles, and ensure that the proteins achieve and maintain their correct shapes and modifications (High tower 1991). Molecular chaperones also are required to transport (i.e., uptake) certain proteins, such as receptors for neurotrans mitters or hormones, from the cell surface into the cells (Chappell et al. 1986).

PROMOTERS AND TRANSCRIPTION FACTORS
Every cell in an organism contains the complete genetic information needed to create that organism. Not all cells, however, express all those genes all the time. For example, liver cells do not need to synthesize the neurotransmit ters that relay signals among nerve cells. Similarly, brain cells have no use for liverspecific enzymes. And even a brain cell does not produce neurotrans mitters continuously. These examples illustrate the requirement for a specific pattern of gene expression in each individual cell. An imbalance in the patterns-for example, the expression of a gene in the wrong cell, at the wrong time, or in the wrong amountcan have disastrous consequences for the organism. Conversely, gene expres sion patterns also must be flexible so that they can be adjusted rapidly to the cell's changing requirements in re sponse to internal processes or envi ronmental influences.
To ensure the coordinated expres sion of genes at the right time and in the right tissues of an organism, regu latory elements have evolved that enable a cell to turn the transcription of each gene on and off, as needed. These "switches," or promoters, are stretches of DNA directly adjacent to the gene sequence itself. Some pro moters represent on/off switches, regulating gene expression by an all ornothing pattern. Other promoters resemble "dimmers," which allow for gradual changes in the level of gene expression depending on the environ mental conditions and the require ments of the cell at any given moment. In addition to controlling the level of gene expression (i.e., the level of transcription), promoters also deter mine the site where transcription be gins. The enzyme that performs the chemical reactions of transcription recognizes a certain nucleotide se quence in the promoter as the tran scription initiation site and binds to that site to begin its work.
Promoters regulate gene expression by providing binding sites for certain proteins, the socalled transcription factors (see figure). Many different transcription factors exist in the cells, and researchers constantly are discovering new ones. The binding of certain tran scription factors to the promoter can activate gene expression by facilitating the access of transcription enzymes to the gene. The binding of other transcription factors to the promoter, however, inhibits transcription by blocking the transcription enzyme from accessing the gene. Some transcription factors are present in the cells at all times; others are synthesized only at specific times during development or under specific environmental condi tions. All promoters contain binding sites for several transcription factors, so that the level of gene expression can be finely tuned through coordinated variation in the kind and abundance of the factors present in the cell at a particular time.
Each transcription factor has a specific sequence of DNA nucleotides that it recognizes in the promoter and to which it binds. For many factors, these sequences are known and have been studied in detail. Researchers have introduced changes (i.e., mutations) into these sequences to study how the mutations affect binding and activity of a tran scription factor. In other approaches, pieces of DNA containing the tran scription factor binding sites are re moved from the promoter to study how these deletions-and the resulting inactivation of specific transcription factors-affect expression of a particu lar gene. Finally, by analyzing the nucleotide sequence of the promoter region of a new gene, researchers frequently can predict which transcrip tion factors are involved in regulating the gene's expression and can make educated guesses about the effects of certain manipulations of the cell or its environment on gene expression.

-Michael F. Miles and Susanne HillerSturmhöfel
Research has shown that alcohol inter feres with the normal transport of proteins through the cell. For example, Tuma and colleagues (1991) found that alcohol inhib ited the uptake of specific receptors from the cell surface into liver cells. This alter ation in the trafficking of receptor proteins could alter livercell function. Increased pro duction of chaperone proteins after extended alcohol exposure, however, may compen sate for these changes by altering the trans port and modification of other proteins. This process could represent a way for cells to adapt to the continued presence of alcohol (i.e., develop tolerance). Similarly, acute exposure of the nervous system to alcohol alters the function of several proteins on the surface of nerve cells that serve as re ceptors for neurotransmitters. Molecular chaperoneinduced changes in the trafficking and processing of neurotransmitter recep tors might enable the brain to adapt to the presence of alcohol.

GTPBinding Proteins
GTP (guanosine triphosphate)binding pro teins (G proteins) are part of the signaling cascade that transmits extracellular signals (e.g., hormone signals) into and within the cell. Through several steps, this process leads to changes in the expression and function of specific genes and proteins in response to the incoming signal. Acute doses of al cohol strongly increase certain cellular re sponses to incoming signals, including the activity of G proteins, a reaction that may be harmful to the cell (Gordon et al. 1992).
Cells contain two major kinds of G pro teins. Stimulatory G protein (G s protein) activates the next enzyme in the signal transmission cascade; inhibitory G protein (G i protein) inhibits the next enzyme. Studies in neural cells found that when the cells were exposed to alcohol for several days, G s levels decreased and G i levels in creased (Charness et al. 1988;Mochly Rosen et al. 1988). Such a modification in Gprotein expression could serve to diminish the cell's response to incoming signals. As a result, the cells would become less sensi tive to a variety of stimuli, thus avoiding the "overreaction" seen in the presence of an acute alcohol dose.

MECHANISMS OF ALCOHOL DEPENDENT REGULATION OF GENE EXPRESSION
Researchers have learned much about alcohol's effects on the body by identify ing genes whose expression is modified by alcohol. Not much is known, however, about the way in which alcohol exerts these effects. Current research efforts are focusing on detailed studies of the struc ture and function of individual genes to determine the sites and mechanisms of alcohol's action.

Alcohol's Effects on Promoters and Transcription Factors
Much of the research on how alcohol affects gene expression focuses on DNA regions adjacent to the genes being regulated, the socalled promoters. Promoters function like switches that determine when, and how strongly, a gene is expressed. These switches are operated by the binding of certain pro teins, the transcription factors, to specific stretches of DNA within the promoter region. (For more information on promoters and transcription factors, see sidebar, p. 241.) To study whether alcohol affects gene expression by interfering with the functions of transcription factors, researchers removed various sections of the promoters of alcohol dependent genes and then examined whether gene expression was still sensitive to alco hol. For example, the promoters of both the TH gene and an alcoholdependent mole cular chaperone, Hsc70, contain short DNA stretches that, if placed in the promoter re gions of genes unresponsive to alcohol, can confer alcoholresponsiveness to these genes. Preliminary studies by Miles and colleagues (1993) found that removing these DNA se quences from the Hsc70 promoter almost completely abolished alcohol's ability to affect Hsc70 expression. Experiments are in progress to identify the transcription factors that bind to the alcoholresponsive promoter regions.

Alcohol's Effects on Signal Transmission Cascades
As mentioned earlier, alcohol affects the activity of G proteins, which play an im portant role in the signal transmission within cells. G proteins regulate a subse quent step in the signal transmission cas cade, the generation of socalled "second messengers." These messengers generally are small molecules that relay signals by modulating the activity of other signaling molecules and by altering the expression of various genes. One second messenger, cyclic adenosine monophosphate (cAMP), con trols the expression of a wide variety of genes, including TH (Roesler et al. 1988).
Previous studies have found that acute alcohol treatment of cells leads to an in crease in cAMP levels; chronic alcohol treatment decreases the level of cAMP in cells. Accordingly, alcoholinduced changes in cAMP levels could alter the expression of many cAMPdependent genes, including TH. So far, however, researchers have not been able to determine unequivocally whether alcohol affects TH gene expression through changes in the cAMP concentration or through other mechanisms.
Another component of a signaling cas cade is protein kinase C (PKC), an enzyme that regulates the function of various pro teins by adding phosphate groups to them. PKC also is known to regulate the expres sion of certain genes. Messing and col leagues (1991) found that the activity of PKC increased in neural cells chronically exposed to alcohol. Thus, alcoholinduced changes in PKC activity could alter the expression of other genes.

FUTURE DIRECTIONS
A growing number of studies using neural cell cultures or brain cells from animals ex posed to alcohol have documented alcohol induced changes in gene expression. The exact roles that these genes play in the adaptation of intact animals to longterm alcohol exposure, however, remain to be determined. Alcoholresponsiveness in intact animals still must be confirmed for many genes that respond to alcohol in cell cultures (see table 1).
Investigators also are initiating innova tive approaches, such as transgenic animals, in which the expression of a specific gene has been either increased or completely shut off, to study the roles of individual genes in the physiological and behavioral conse quences of acute and chronic alcohol use (Harris et al. 1995). 3 If researchers are able to identify individual genes linked to toler ance, these findings may have important consequences. By studying the function and mechanisms involved in the regulation of these genes, researchers may someday generate new treatments for alcoholism. A drug that interferes with alcoholdependent regulation of gene expression, for example, might aid other treatments attempting to reduce the longterm alcoholseeking be havior associated with alcoholism. ■